Conformational effects on the Circular Dichroism of Human Carbonic Anhydrase II: a multilevel computational study

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions

Mahepõllumajanduse alused

TäistekstMahe- ehk ökoloogiline põllumajandus on loodushoidlik tootmisviis, mis põhineb tasakaalustatud aineringlusel ja kohalikel taastuvatel varudel. MAHEPÕLLUMAJANDUSE PÕHIMÕTE ON TOIMIDA KOOS LOODUSEGA, MITTE TEMA ARVEL. Eduka maheviljeluse eelduseks on arusaam, et toidu tootmine on pigem ökoloogiline kui tehnoloogiline protsess. Põllumajandus erineb juba oma olemuselt teistest tootmisharudest, olles otseselt ökosüsteemi osa. Tavapõllumajanduses püütakse agroökosüsteeme võimalikult lihtsaks muuta. Rohkem kui viiekümne aasta jooksul on enamikus Euroopa riikides nii põllumajanduspoliitika, -hariduse kui ka nõustamise kaudu talunikele sisendatud, et tuleb tootmist intensiivistada ja võimalikult kitsalt spetsialiseeruda. Teisisõnu, talunikke on tootlikkuse huvides ärgitatud vähendama oma karja ja kultuuride mitmekesisust ning laiendama intensiivtehnoloogiate kasutuselevõttu. Mahepõllumajanduses lähtutakse aga ökoloogilisest printsiibist, et mida keerukam ja mitmekesisem on agroökosüsteem, seda stabiilsem ta on. Näiteks tekitab haiguste või kahjurite rünnak monokultuuri puhul alati suuremat kahju. Seetõttu kasvatatakse paljusid eri kultuure, säilitatakse looduslikke alasid ja soodustatakse mitmekesise elustikuga servaalade teket. Teades, et paljud agroökosüsteemides toimivad protsessid on tootmisele kasulikud, püütakse neid võimalikult efektiivselt ära kasutada, mitte aga ignoreerida või alla suruda. Et mahetootmises on sünteetilised pestitsiidid ja mineraalväetised välistatud, siis peab mahetootja loodust rohkem tundma kui tavatootja ning oma tegevuse palju põhjalikumalt läbi mõtlema. Tuleb leida kohalikesse oludesse sobiv külvikord, sobivad liblikõielised, vahekultuurid, harimistööde aeg, sõnniku säilitamise viis, selle põllule andmise aeg jne. Mahepõllumajandus ei tähenda pöördumist minevikku. See on traditsiooniliste põllumajandusmeetodite ning uusima teadusliku ja tehnoloogilise teabe kombinatsioo

Lack of correspondence between the room-temperature phosphorescence decay-components and Trp residues in a series of Trp-->Cys or Trp-->Phemutants of human carbonic anhydrase II

The room temperature phosphorescence of native human carbonic anhydrase (CA), and several mutants of this enzyme has been investigated. In these mutants the seven tryptophan residues in the native protein have sequentially been replaced by cysteine or phenylalanine. All of the mutants as well as native CA show room-temperature tryptophan phosphorescence (RTP) spectra. Surprisingly, only small differences in RTP life-times are noticeable among these mutants, indicating that there is more than one tryptophan residue with similar phosphorescence decay kinetics in the protein. The present results illustrate the danger in attributing the room temperature phosphorescence of a multi-tryptophan protein to a particular residue based solely on an analysis of the protein structure.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31251/1/0000157.pd

Effects on the conformation of FVIIa by sTF and Ca(2+) binding : Studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent  fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.Funding agencies|Swedish Research Council||Knut and Alice Wallenbergs Foundation|

resonance energy transfer and quenching.

Effects on the conformation of FVIIa by sT